This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Smallpox is a threat in an event of a bioterrorist attack, therefore it is important to develop a safe vaccine against this virus. There is an important population of non-vaccinated immunocompromised people that could not be safely vaccinated with the current vaccine. Within these are patients of transplants, under immunosupressive therapy, pregnant women, and the elderly. We are interested in the development of a safer vaccine that protects the population against a possible attack. We hypothesize that the adjuvant-mediated enhancement of the molecular antigen immune response will control the viral burden on infected mice. However, as smallpox is only one of possible threats, this project will provide an excellent model to test the hypothesis of a molecular adjuvant that will help set up a system that could be applied to other Category A, B and C Priority Pathogens. Mice will be immunized with plasmid DNA encoding for the vaccinia virus proteins C7L and A55R genes that are non structural proteins known to generate a cellular immune response, and the recently discovered E6R and L3L core proteins. The immunology of those molecules has not been tested to the best of our knowledge. Animals will be vaccinated with each construct individually or combined with two molecular adjuvants, the chemokine MIP-1-alpha and another construct which encodes for the dendritic cell-specific growth factor Flt-3L, a combination that is known to increase the recruitment and expansion of dendritic cells. To the best of our knowledge, this combination of molecular adjuvants has never been tested in the context of smallpox vaccination. Cell- and humoral-mediated immune responses from each antigen will be determined by ELISPOT and ELISA, respectively. Molecular mapping of each antigen will be performed to identify dominant epitopes. Animals will be challenged with a lethal dose of vaccinia virus, and the viral load will be determined by qPCR analysis. Memory, activation and proliferation will be determined to detect the long-lasting effect of each antigen on the induction of an immune response. Strong cellular and humoral immune responses against vaccinia virus are expected so that protecting immune memory could be developed without the hazard of being exposed to live virus.